The Analytical module of FELIX assists chemists in resonance assignment for small molecules. It includes controls for structure display, spin system detection, and manual assignment of 1D/2D peaks to atoms.
In Lesson 1: Resonance assignment of brucine, you will learn the basic steps involved in assigning a natural product using conventional 2D spectra.
This lesson presents the basic steps of an NMR spectrum assignment using prepared 1D and 2D data: the H-1, DEPT-135, TOCSY, and HMQC spectra, of brucine (MF: C23H26N2O4) and its structural diagram prepared with ISIS/Draw).
The topics covered in this lesson are:
1. Setting up for the lesson
2. Starting FELIX
3. Going to the Analytical module
4. Setting up the database
This procedure typically takes several seconds.
5. Viewing the project entity through a spreadsheet
The project entity is presented in a spreadsheet, and you can browse through its fields.
Many fields contain zeros or nulls, since the project is not fully defined yet. Also note that there are twelve experiment columns, meaning that you can define twelve experiments in one project.
6. Adding the 1D H-1 experiment to the project
Two ways of adding experiments to a project are shown in Steps 6-9 of this lesson.
Select File/Open from the main menu. In the OPEN FILE control panel, select FELIX New Data (*.dat) as the File Type. When the list of names of 1D spectra in the current directory appears, select br_h1.dat (the H1 spectrum). Select OK.
This adds the currently displayed H1 experiment to the project.
This adds the H1 experiment to the project with the current display parameters. You can change the display parameters using the Experiment/Change Attribute menu item in the Experiments table (select the Analytical/Experiments menu item to display the Experiments table).
7. Adding the DEPT-135 experiment to the project
Select the Analytical/Experiment... item in the main menu to open the Analytical Experiments table. In the table, make sure that one row is highlighted (otherwise it will not work) and select the Experiment/Add menu item.
Select the File/Import/Peaks... item from the main menu. In the control panel, make sure that FELIX Peak Table Name is pic:dept. When the list appears with names of the peak files (.txt) in the ./ directory, select br_dept.txt (the DEPT peak table). Select OK to import the DEPT peaks.
The peaks are labelled on the DEPT spectrum. The C13 peaks are also
listed in the spreadsheet table Peaks - pic:dept.
This demonstrates two ways of adding experiments to a project: you can add the currently displayed experiment or a saved experiment.
8. Repeating Step 6 for the HMQC experiment
Select the File/Open item from the main menu. In the OPEN FILE control panel, select Matrix (*.mat) as File Type. When the list appears with matrix names of 2D spectra in the ./ directory, select br_hmqc.mat (the HMQC spectrum). Select OK.
Adjust the display of the spectrum until it is satisfactory.
Select the File/Import/Peaks... item from the main menu. Make sure that FELIX Peak Table Name is xpk:hmqc. When the list appears with names of the peak files (.txt) in the ./ directory, select br_hmqc.txt (the HMQC peak file). Select OK to import the HMQC peaks.
The peaks are labelled on the HMQC spectrum. The HMQC peaks are also listed in the spreadsheet table Peaks-xpk:hmqc.
This adds the currently displayed HMQC experiment to the project.
The HMQC spectrum is added to the project.
9. Repeating Step 7 for the TOCSY spectra
If it is not open, select Analytical/Experiments to open it.
This opens a TOCSY spectrum.
In the 2D DISPLAY PARAMETERS control panel, click the Set button if you find the display satisfactory. Otherwise you can click the Full button to display the full spectrum or the Zoom button to zoom in on a certain spectral region. If the display is still not satisfactory, click No in the GENERAL MESSAGE control panel to return to the 2D DISPLAY PARAMETERS control panel.
If the HMQC peak labels are displayed on the spectrum, ignore them for
Select the File/Import/Peaks... item from the main menu. Make sure that FELIX Peak Table Name is xpk:tocsy. When the list appears with names of the peak files (.txt) in the ./ directory, select br_tocsy.txt (the TOCSY peak table). Select OK.
This imports the TOCSY peaks.
The peaks are labelled on the TOCSY spectrum. The peaks are also listed in the spreadsheet table Peaks-xpk:tocsy.
10. Checking the project entity
Check the project entity after all experiments are added, as described in Step 5.
The previously zero or null fields now have values.
11. Drawing the full HMQC spectrum
12. Performing prototype pattern detection
Select the Analytical/Collect Prototype Patterns menu item. From the control panel, select the HMQC+TOCSY as the Method (the default value) and make sure that tocsy and hmqc are selected for TOCSY Experiment and HMQC Experiment, respectively. Enter 0.012 as the Interspectral Tolerance for H. Select OK.
Information about the current stage of prototype pattern collection is displayed in the text window. The prototype pattern collection finishes quickly, and the following information appears in the text window:
Nr of prototype patterns generated:(8)
The 2D protopattern detection took 1 seconds
Also, a spreadsheet containing the 8 prototype patterns is displayed (Protopatterns table).
13. Writing the result of prototype pattern detection into a file
The text window shows information about the success of the action:
Wrote table: br:proto
Created file: ./br_protos.txt
14. Visualizing prototype patterns
The next step is to visually inspect the prototype patterns. Several ways
of seeing prototype patterns are provided through the Protopatterns
spreadsheet: you can draw frequencies of prototype patterns as lines on
top of a contour plot, spawn tiles, or draw a strip plot.
On the HMQC spectrum, you see six lines on D1, and 12 lines on D2 if it is a full plot. Among the 12 lines on D2, only six are C13 lines, while the remaining six are the symmetrical images of the D1 H1 lines. You can recognize them based on whether they are correlated to any HMQC peaks.
You can also choose to display the TOCSY spectrum and then plot the
prototype pattern on it.
This clears the frequency lines on the spectrum.
15. Making a tile plot of prototype pattern
The second way to visualize prototype patterns is to spawn tile plots from them. This allows you to concentrate only on frequencies and peaks belonging to them, which are present in this prototype pattern.
If you want to change the tile plot attributes, select the Preferences/Tile Plot menu item from the table.
The tile plot is displayed.
If you want to switch back to intensity plot mode, press <Ctrl>-i.
You can also display frequencies by clicking the Draw icon in the table.
16. Making a strip plot of a prototype pattern
Using the tile plot functionality, you can concentrate on peaks and their
immediate surroundings which belong to a prototype pattern. You can
also use strip plots to see strips surrounding the frequencies in vertical
or horizontal orientations.
This action (Jump) places only that small region on the screen and returns from tile plot mode.
You see the seven HMQC peaks relevant to the frequencies of the third prototype pattern displayed in horizontal strips. You can also display the frequencies by selecting the Draw icon.
The strip plot helps you verify if there are outstanding peaks that have common chemical shifts with the frequencies in this prototype pattern.
17. Copying a prototype pattern to the frequency clipboard
In practice, because of peak overlap or missing peaks, the automatically
detected prototype patterns may have wrong connectivities, which
need to be manually corrected. FELIX provides a set of tools to verify
and edit the prototype patterns. These actions can be accessed from the
pullright menu by selecting Analytical/Frequency. The first step in this
procedure is to copy the frequencies of a certain prototype pattern to the
Detach the Frequency Clipboard item from the menu bar by selecting the Analytical/Frequency Clipboard item from the top row of the pullright menu. Place the Frequency Clipboard menu in a convenient location.
The third prototype pattern is now copied to the clipboard list. This list can be manipulated (you can add or delete frequencies to or from the list, swap the order of two frequencies, delete duplicate frequencies, sort the list, or zero the list). You can also display the list as lines on top of the matrix plot and spawn a tile and strip plot from it.
Select the Sort Clipboard menu item in the Frequency Clipboard menu. Now you can sort the frequencies in the clipboard in descending ppm order by toggling Descending order to on and then selecting OK.
You can see the sorted clipboard by selecting the View Clipboard item from the Frequency Clipboard menu. The result should look like this:
The Frequency Clipboard List contains the following fre- quencies:
# Freq(ppm) Atom
--- --------- ----
1 127.315 Y
2 64.597 Y
3 60.076 Y
4 56.463 Y
5 31.469 Y
6 26.774 Y
7 5.881 X
8 4.099 X
9 3.851 X
10 3.139 X
11 2.347 X
12 1.456 X
18. Drawing the clipboard prototype pattern
As described in Step 11, you can use the Experiment Table to display the HMQC or TOCSY spectrum before viewing the prototype pattern.
The spind system is displayed on the 2D spectrum.
Similarly, you can select Tile Clipboard or Strip Plot Clipboard menu item from the Frequency Clipboard menu to display the clipboard spin system in tile or strip view. These tools are useful when you edit the prototype pattern in the clipboard, as shown in the next step.
19. Editing the clipboard prototype patterns
If you find it necessary to edit the prototype pattern in the clipboard,
choose the Add One, Delete One, Swap Two, or Remove Duplicates
menu item in the Frequency Clipboard menu to add, remove, swap, or
purge frequencies in the prototype pattern.
After you are finished editing, select the Copy Clipboard To Proto item from the Frequency Clipboard menu. In the control panel that appears, set Prototype Pattern to 3 and Overwrite to Mode. Then select OK,
The third prototype pattern in the Protopatterns table is updated.
20. Import and display molecular structure
The prototype patterns usually give you an idea of the assignment of the frequencies to individual atoms in the molecule. Once you are ready to assign the resonances, you can input the chemical structure and interactively assign the resonances in a 1D or 2D peak table to the atoms.
The structure is displayed with atom labels on.
You can also rotate, translate, or reset the display by choosing options in the 1 Player control panel.
Since this is a planar structure (i.e., all Z coordinates are zero), you can
only rotate it in the plane of the screen.
You can select the Analytical/Draw Molecule, Analytical/Label..., and Analytical/Color... menu items to redraw the molecule, change the display of labels, or change the colors if necessary.
Currently, all bonds are displayed as single lines. The carbonyl group C1=O19, double bond C1=C13, and phenyl ring consisting of C20- C25 are not displayed as such.
21. Assigning 1D DEPT peaks to atoms
Now there are two empty frames.
If it is not open, select Analytical/Experiment... to open it.
The DEPT spectrum is displayed in the new frame. If the DEPT peak
labels are not displayed, toggle on View/Draw Peaks.
The values in the name column are all null, which means that the corresponding
peaks are not assigned yet.
From the chemical shift it obviously belongs to the only carbonyl carbon c1.
Notice that the name cell of peak 1 is now c1.
If you want to unassign a peak, highlight it and select Peak/Unassign One. The name of the peak is restored to null.
Once you are done with the assignment of the DEPT peak table, select File/Save As from the Peaks-pic:dept table to export the peak table with assignments. In the control panel, enter a filename, such as deptassign.txt, and select OK.
The DEPT assignments are stored in an ASCII file.
22. Assigning the HMQC peaks to atoms
You can also assign the C13 frequencies in the HMQC peak table to the carbon atoms. Note that quaternary carbons are not present in the peak table, and that since hydrogen atoms are not explicitly displayed in the structure, you cannot assign the H1 frequencies to hydrogens.
If it is not open, select Analytical/Experiment... to open it.
The HMQC peaks are displayed in a Peaks-xpk:hmqc table. Notice that the column NameD1 and NameD2 are all nulls, which means that the corresponding frequencies are not assigned yet.
From the chemical shift it obviously belongs to the sp2 methine carbon C11. (Remember that double bonds are not displayed.)
You'll notice that the name cell of this frequency changes to C11.
If you want to unassign a frequency, highlight it (either the chemical shift or the name) and select Peak/Unassign One. The name of the peak is restored to null.
Once you are done with assignment of the HMQC peak table, select File/Save As from the Peaks-xpk:hmqc table to export the peak table with assignments. In the control panel, enter a filename, such as hmqcassign.txt, and select OK.
The HMQC assignments are stored in an ASCII file.
23. Exiting FELIX